Mechanistic insights into the C55-P targeting lipopeptide antibiotics revealed by structure–activity studies and high-resolution crystal structures

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.


Reagents and General Methods
All reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. D-amino acids and 2-chlorotrityl resin was obtained from Iris Biotech GmbH, Egg PG and 0:6 PA was obtained from INstruchemie BV. C 10 -P lithium salt was obtained from Sigma Aldrich and lyophilized from warm t BuOH:H 2 O (1:1) to obtain a white powder with increased aqueous solubility.

Instrumentation for Compound Characterization
2D NMR experiments were performed on a 850 MHz instrument. HSQC, TOCSY and NOESY spectra were recorded for all peptides (5 mM in DMSO d6 ) and the parent compound laspartomycin C matched pervious recorded spectra reported by our group.
HRMS analysis was performed on a Shimadzu Nexera X2 UHPLC system with a Waters Acquity HSS

S4
The compounds were purified via preparative HPLC using a BESTA-
Chloramphenicol (130 µg mL -1 ) was added and after incubation for 15 minutes at 37°C, the culture was divided in 5 mL aliquots. Antibiotics were added at 10xMIC and one aliquot remained untreated. After 60 minutes, cells were separated from the medium and extracted with boiling d-H 2 O (1 mL) for 15 minutes.
The suspensions were spun down and the supernatant was lyophilized. The resulting material was analyzed by HPLC applying a gradient from 100% eluent A (50 mM NaHCO 3

Structure solution and refinement
The dataset of lipopeptide 5 was processed in the DIALS pipeline 2 , whereas autoPROC 3 was used for lipopeptide 7. The crystal of lipopeptide 7 was initially indexed in a hexagonal setting but based on the merging R-values the true symmetry appeared to be Primitive monoclinic with β = 120°. The reflection file was therefore re-indexed accordingly, and parameters for pseudo-merohedral twinning were included in the structure refinement. Additional anisotropic correction was done for the datasets of both analogues in STARANISO. 3 Structures were solved by molecular replacement using PHASER, 4 and one copy (lipopeptide 5) or one dimer (lipopeptide 7) of laspartomycin C in complex with geranyl phosphate (PDB: 5O0Z) 5 was used as a search model. Models were manually improved in Coot, 6 Figure S2. A) Structure of the ternary complex with lipopeptide 7 (green stick representation), two bound Ca 2+ ions (orange spheres), a bound water molecule (red sphere), and the C10-P ligand (lipid in grey). B) Lipopeptide 7 adopts a saddle-shaped conformation when complexed with two Ca 2+ ions and C10-P and forms a dimer in the crystal.  5 Asymmetric units are composed of one monomer for lipopeptide 5 (the dimer shown for lipopeptide 5 generated by applying two-fold crystallographic symmetry), six dimers for lipopeptide 7 and one dimer for laspartomycin C. RMSD between dimers is indicated in the table. Ca 2+ ions are represented by orange spheres and water molecules are represented by red spheres, C10-P is also indicated. Figure S4. In the crystal state lipopeptide 5 forms a higher-ordered assembly when complexed with Ca 2+ and C10-P consisting of alternating hydrophobic (grey), peptidic (green), and hydrophilic (red) layers. A similar lattice is also observed for lipopeptide 7. Notably, this higher ordered assembly is not seen for laspartomycin C.
Fluorescence microscopy was carried out using a Zeiss Axiovert 200M equipped with a Zeiss Neofluar 100x/1.30 Oil Ph3 objective, a Lambda S light source (Shutter Instruments), a Photometrics Coolnap HQ2 camera, and Metamorph 6 software (Molecular Devices). Images were analyzed using ImageJ (National Institutes of Health) v.1.52a.